HerdiniIndonesian et al. Journal of Biotechnology, June, 2015 Vol. 20, No.I.J. 1, pp.34-41Biotech. Diversity of Nonribosomal Peptide Synthetase Genes in the Anticancer- Producing Actinomycetes Isolated from Marine Sediment in Indonesia

Camelia Herdini1,5, Shinta Hartanto1, Sofi a Mubarika1, Bambang Hariwiyanto1,5, Nastiti Wijayanti1, Akira Hosoyama2, Atsushi Yamazoe2, Hideaki Nojiri3, Jaka Widada4

1 Graduate School of Biotechnology, Universitas Gadjah Mada, Barek Utara, Yogyakarta, Indonesia 2 Biological Resource Center, National Institute of Technology and Evaluation, Nishihara, Shibuya-ku, Tokyo, Japan 3 Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 4 Department of Agricultural Microbiology, Universitas Gadjah Mada, Bulaksumur, Yogyakarta, Indonesia 5 ORL Head and Neck Department, Faculty of Medicine, Universitas Gadjah Mada, Sekip, Yogyakarta, Indonesia

Abstract Marine actinomycetes is a group of bacteria that is highly potential in producing novel bioactive compound. It has unique characteristics and is different from other terrestrial ones. Extreme environmental condition is suspected to lead marine actinomycetes produce different types of bioactive compound found previously. The aim of this study was to explore the presence and diversity of NRPS genes in 14 anticancer-producing actinomycetes isolated from marine sediment in Indonesia. PCR amplifi cation and restriction fragment analysis of NRPS genes with HaeIII from 14 marine actinomycetes were done to assess the diversity of NRPS genes. Genome mining of one species of marine actinomycetes (strain GMY01) also was employed towards this goal. The result showed that NRPS gene sequence diversity in 14 marine actinomycetes could be divided into 4 groups based on NRPS gene restriction patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that all isolates belong to genus of Streptomyces. Genome mining result showed that strain GMY01 harboring 10 different NRPS gene clusters that encode secondary metabolites, as pure NRPS or hybrid between NRPS and other compounds. These results indicated that marine actinomycetes having a high potential to be developed as source of anticancer drugs development.

Keyword: marine actinomycetes, non-ribosomal peptide synthetase genes diversity, Streptomyces, genome mining, anticancer drugs development

Introduction actinomycetes decreased, whereas the rate of Wide range of bioactive compounds re-isolation of known compounds increased metabolites were isolated and identified (Dharmaraj, 2010). Thus, it is crucial that new from soil actinomycetes. Recently, the rate group of actinomycetes from unexplored of new metabolites discovery from terrestrial habitats to be pursued for new sources of bioactive compounds, one of which is marine actinomycetes (Magarvey et al., 2004). * Corresponding author: Nonribosomal peptide synthetases Camelia Herdini (NRPS) and type I polyketide synthases Graduate School of Biotechnology, Universitas (PKS-I) are biosynthetic systems involved in Gadjah Mada, Barek Utara, Yogyakarta, Indonesia. Email: [email protected] or Jaka Widada the synthesis of a large number of important [email protected]) biologically active compounds produced

34 Herdini et al. I.J. Biotech. by microorganisms, among others by Material and Methods actinomycetes (Ayuso-Sacido and Genilloud, Bacterial strains and culture conditions. 2004). The 14 anticancer-producing Farida et al. (2007) isolated several actinomycetes isolated from marine marine actinomycetes from Krakal Beach, sediment in Indonesia used in this study Gunung Kidul, Yogyakarta and proved the were from previously study (Farida et al., anticancer effects of the marine actinomycetes 2007). All strains used in this study were extracts. That research results also showed grown in shaking fl asks containing starch that the anticancer compounds from marine nitrate broth with fi ltered seawater at room actinomycetes suggested maybe belong to temperature. All strains were stored at -80°C the poliketide or non-ribosomal peptides as suspensions of spores and hyphae in 15 % based on the presence of NRPS or PKS gene. (v/v) glycerol. According to Wang et al. (2014), most of the screening methods performed were to explore Isolation of chromosomal DNA a new bioactive compounds that focusing on Chromosomal DNAs were isolated by the detection of NRPS or PKS genes. NRPS a versatile quick-prep method for genomic gene is one of the genes involved in the DNA of Gram-positive bacteria (Pospiech and synthesis of a variety of important bioactive Neumann, 1995), with some modifications. compounds called non-ribosomal peptides. Bacterial culture (1 ml) grown in a starch nitrate Many researchers have made isolation broth shake culture were centrifuged, rinsed process of bioactive compounds from with TE and re-suspended in 0.4 ml TE buffer. terrestrial actinomycetes, but after the Lysozyme-1 was added to a concentration of 1 elucidation process, bioactive compounds mg ml and incubated at 37°C for 1 h. Then 0.1 were often structurally similar to the structure vol 10 % SDS was added and incubated at 65°C of bioactive compounds that was found with occasional inversion for 2 h. One-third previously, so information about NRPS genes volume 5 M NaCl and 1 vol. chloroform were sequence and diversity in actinomycetes added and incubated at room temperature for are very important to avoid the risk of re- 0.5 h with frequent inversion. The mixture was isolation. This approach can be used as a tool centrifuged at 13,000 rpm for 10 min and the for initial screening in the discovery of new aqueous phase was transferred to a new tube bioactive compounds from actinomycetes. using a blunt-ended pipette tip. Chromosomal Referring to research conducted by Farida DNA was precipitated by the addition of 1 vol. et al. (2007), although marine actinomycetes 2-propanol with gentle inversion. The DNA that could potentially have anti-cancer was transferred to a new tube, rinsed with 70 % compounds and detected carry NRPS gene ethanol, dried under vacuum and dissolved in in the genome, but these studies have not a suitable volume (about 50 ml) of TE buffer. observed NRPS genes sequence and their diversity. NRPS gene PCR Amplification and The aim of this study was to explore Restriction Endonuclease Digestion the presence and diversity of NRPS genes in The NRPS gene was amplified using 14 marine actinomycetes. PCR amplifi cation primers A3 (5’GCSTACSYSATSTACA and restriction fragment analysis of NRPS CSTCSGG’3) and A7R (5’SASGTCVCCSGTS genes from different species of marine CGGTAS ‘3) (Ayuso-Sacido and Genilloud, actinomycetes were done. Genome mining 2004). Amplifi cation of the NRPS gene was of one species of marine actinomycetes (strain performed in T100 thermal cycler (Bio-Rad) GMY01) also was employed towards this in a total volume of 50 ml containing 30-50 ng goal. DNA, 100 mM each primer, 10 mM dNTP, 10X buffer and 1.5 U Taq DNA polymerase

35 Herdini et al. I.J. Biotech.

(Promega). PCR program used to follow (NGS) using 454 pyrosequencing technology the procedures performed by Farida et al. (454 GS FLX) and HiSeq1000 (Illumina) (2007) with modifications. PCR program (Herdini et al., unpublished). Genome mining included initial denaturation at 97° C for 3 min, and analysis of gene cluster involved in the denaturation at 95°C for 1 min, then followed by biosynthesis of secondary metabolites of annealing at 55°C for 1 min, extension at 72°C Streptomyces sp. GMY01, especially gene for 3 min the whole process takes place as many clusters containing NRPS, were done with as 30 cycles, the last cycle followed by a fi nal antiSMASH 3.0 (Medema et al., 2011; Weber extension at 72°C for 10 min and cooling stage et al., 2015) available on http://antismash. at 4°C. The PCR product was visualized by 1% secondarymetabolites.org. agarose electrophoresis. Restriction enzyme, HaeIII, was used to digest the amplifi ed NRPS Data analysis genes fragment. The reactions were performed Data subjected from electrophoresis in fi nal volumes of 20 μl containing at least 8 were analyzed using UPGMA method. μg∙ml-1 of NRPS genes products at 37°C for 3 h. The sequences of 16S rRNA determined in The digestions were then analyzed by agarose this study were aligned using CLUSTAL gel electrophoresis (2%, w/v) for 1 h at 110 W software (Thompson et al., 1994). The V followed by visualization of the banding nucleotide similarity values were calculated patterns using a UV transilluminator (UVP from the alignment. Evolutionary trees for the Canada). datasets were inferred from the neighbour- joining method (Saitou and Nei, 1987) using PCR Amplifi cation and sequencing of 16S MEGA version 6.0 (Tamura et al., 2013). The rRNA genes stability of relationships was assessed by The 16S rRNA gene was amplified performing bootstrap analysis of neighbour- using primers 27F (5’-AGA GTT TGA TCC joining data based on 1,000 resampling. TGG CTC AG-3 ‘) and1492 R (5’-GTT TAC CTT GTT ACG ACT T-3 ‘) (Jiang et al., 2007). Result and Discussion Amplification of the 16S rRNA gene was Marine Sediment-derived Actinomycetes performed in T100 thermal cycler (Bio-Rad) in Fourteen actinomycetes that isolated a total volume of 50 ml as a described above. from marine sediments in Krakal beach in PCR program used to follow Jiang et al. (2007) Indonesia and produce anticancer (Farida et with some modifi cations, the PCR program al., 2007) were used in this study. Based on included initial denaturation at 97°C for 3 min, colony morphology after 14 days of growth denaturation at 95°C for 1 min, then followed on solid medium starch nitrate at 30°C showed by annealing at 60°C for 1 min, extension at that all 14 actinomycetes diverse and different 72°C for 3 min the whole process lasted about from each other (Figure 1). From the colony 30 cycles, the last cycle followed by a final morphology also indicates that almost all extension at 72° C for 10 min and cooling stage isolates are most likely the genus Streptomyces. at 4°C. The PCR product was visualized by 1% It is known that the genus of Streptomyces is a agarose electrophoresis. The 16S rRNA gene group of actinomycetes that generate the most was sequenced with primers designed by Chun bioactive compounds, including anticancer and Goodfellow (1995). (Newman et al., 2000; Doroghazi et al. 2014).

Genome Mining of Streptomyces sp. Diversity of Nonribosomal Peptide GMY01 Synthetase Genes Previously, sequence analysis of the Nonribosomal peptide synthetase genome of Streptomyces sp. GMY01 was done (NRPS) gene in 14 actinomycetes from with platforms Next Generation Sequencing marine sediments amplifi ed using primers,

36 Herdini et al. I.J. Biotech.

Figure 1. Morphology of 14 anticancer-producing actinomycetes isolated from marine sediment in Indonesia after 14 days of growth on starch nitrate solid medium at 30°C. Streptomyces lavendulae was included as a comparison

700 bp

Figure 2. Visualization of NRPS gene amplifi cation of 14 marine actinomycetes isolated by electrophoresis in 1% (w/v) agarose gel stained with etidium bromide (above), and HaeIII restriction patterns of NRPS gene 14 marine actinomycetes isolated by electrophoresis in 2% (w/v) agarose gel stained with etidium bromide (bottom). Lanes 1: GMY 1, 2: GMY 2, 3: GMY 15, 4: GMY 16, 5: GMY 23, 6: GMY 29, 7: GMY 6, 8: GMY 9, 9: GMY 4, 10: GMY 41, 11: GMY 55, 12: GMY 56, 13: GMY 57, 14: GMY 50. Using marker 100 bp DNA ladder.

A3 and A7, which had been previously were generally varied (700-800bp) (Ayuso- developed and used by Ayuso-Sacido et Sacido and Genilloud, 2004; Ayuso-Sacido al. (2004). Primary A3 and A7R used to et al., 2005). strengthen adenilation NRPS gene domain To determine the diversity of NRPS with a targeted size of approximately 700- genes, restriction enzyme analysis with 800 bp (Figure 2A). Figure 2A shows that HaeIII enzyme was done, and the result was the sizes of the PCR products of NRPS all presented in Figure 2B. The result showed that isolates were almost identical at 700 bp. This HaeIII could be used to classify the NRPS gene result was in contrast with previous studies diversity. HaeIII enzyme restriction was used that the sizes of the PCR products of NRPS to cut the NRPS genes because actinomycetes

37 Herdini et al. I.J. Biotech.

GMY1 - I GMY41 GMY4 GMY55 GMY56 II GMY57 GMY50 GMY9

GMY16 III GMY15 GMY29 GMY2 IV GMY6 GMY23

0.4 0.5 0.6 0.7 0.8 0.9 1 Figure 3. UPGMA dendrogram shows the clustering of 14 actinomycetes isolates generated from amplifi ed NRPS genes restriction analysis with restriction endonuclease HaeIII, using the UPGMA algorithm and the Jaccard’s coeffi cient. The Roman numerals I to IV represent the four clusters of NRPS obtained in the analysis. The isolates subjected to 16S rRNA gene sequencing analysis are highlighted in boldface

GMY57 Streptomyces plicatus strain NBRC 13071(2) Streptomyces geysiriensis strain NBRC 15413 Streptomyces rochei strain NRRL B-1559 Streptomyces vinaceusdrappus strain NRRL 2363 GMY29 Streptomyces mutabiis strain NRRL ISP-5169 Streptomyces naganishii strain NRRL B1816 Streptomyces djakartensis strain NBRC 15409 Streptomyces tendae strain ATCC 19812 Streptomyces griseofl avus strain LMG 19344 Streptomyces variabilis strain NRRL B-3984 Streptomyces griseorubens strain NBRC 12780 GMY16 Streptomyces fumigatiscleroticus strain NBRC 12999 Streptomyces mexicanus strain NBRC 100915 Streptomyces chromofuscus strain NBRC 12851 Streptomyces deseri strain C63 16S GMY01 Kitasatospora setae KM-6054

0.0050 Figure 4. Neighbour-joining tree based on partial 16S rRNA gene sequences, showing relationships between GMY01, GMY16, GMY29 and GMY57 and closely related to members of the genus Streptomyces. The numbers at the nodes indicated the level of bootstrap support (%) based on a neighbour-joining analysis of 1.000 resampled datasets, only values above 50% are given. has DNA with a high G and C bases content, at a G and C rich region. Enzyme HaeIII the content of G and C bases in the genome restriction endonucleases are enzymes which of actinomycetes are high between 56-64% have a tetra metric site restriction to the four (Dharmaraj, 2010). HaeIII enzyme cuts DNA bases, this enzyme cut the DNA sequences

38 Herdini et al. I.J. Biotech.

Table 1. NRPS gene cluster of secondary metabolites from Streptomyces sp. strain GMY01 based on AntiSMASH analysis

No. Type of gene cluster Size (bp) Most similar known cluster (%) MIBiG BGC-ID 1 Nrps 43303 Stenothricin (13) BGC0000431_c1 2 Nrps 51715 - - 3 Nrps 91515 Eduracidin (12) BGC0000341_c1 4 Nrps 68122 Mirubactin (50) BGC0000392_c1 5 Nrps 62061 Scabichelin (90) BGC0000423_c1 6 Nrps 16929 Naphthyridinomycin (21) BGC0000394_c1 7 T1pks-Butyrolactone-Nrps 122282 Rifamycin (38) BGC0000137_c1 8 Nrps-T1pks 29889 SGR_PTMs (50) BGC0001043_c1 9 Bacteriocin-Nrps 51659 Griseobactin (47) BGC0000368_c1 10 Lantipeptide-Nrps 67317 Rifamycin (9) BGC0000136_c1 in regions that have GGCC base sequences. GMY16, GMY29 GMY57 and closely Other restriction enzymes can cut the NRPS related members of the genus Streptomyces. gene and other genes in actinomycetes such Neighbour-joining tree based on partial 16S as HinfI can cut NRPS genes, PKS I and II rRNA gene sequences showed that GMY01, produced a diverse banding pattern (Ayuso- GMY16, GMY29 and GMY57 are close Sacido et al., 2005), while the enzyme MspI related to with S. deserti, S. griseorubens, S. and TaqI can cut NRPS gene and 16S rRNA vinaceusdrappus, and S. rochei respectively. in actinomycetes (Jiang et al., 2007). Based In this study, restriction fragment of NRPS on the NRPS gene cutting pattern of 14 gene with HaeIII enzyme, followed by isolates of marine actinomycetes, restriction analysis of 16S rRNA gene sequences of analysis with restriction endonuclease HaeIII representatives from each group have of NRPS genes can be used to analyzed the provided preliminary information that diversity of NRPS gene sequence among 14 14 anticancer-producing actinomycetes actinomycetes isolates quickly. isolated from marine sediment in Indonesia Cluster analysis of restriction pattern (Farida et al., 2007) have high diversity of of NRPS genes using the UPGMA algorithm NRPS genes sequence and have the potential and the Jaccard’s coeffi cient was showed in for producing other important bioactive Figure 3. From that figure, there were four compounds. groups of actinomycetes based on NRPS Genome mining of GMY01 by using genes, which have the coeffi cient of 1. All four AntiSMASH showed that GMY01 harboring groups are group 1 (GMY01); group 2 (GMY04, 6 NRPS genes and 4 hybrid NRPS genes, GMY09, GMY41, GMY55, GMY56, GMY50 and with sequence identities to the most similar GMY57); group 3 (GMY15 and GMY16); and known cluster range from 9 to 90% (Table 1). group 4 (GMY02, GMY06, GMY23 and GMY This result indicated that each Streptomyces 29). It means that the isolated groups were harbor diverse NRPS genes. expected to have high sequence similarity on Our strategy to identify NRPS genes adenilation domain of NRPS gene. Based on within 14 marine actinomycetes using the dendogram in Figure 3, four isolates were two different approaches resulted in selected for sequencing analysis of 16S rRNA new findings. By in silico data mining of gene; they were GMY01, GMY16, GMY29 and GMY01, we gained a valuable insight into GMY57. the abundance and origin of NRPS genes Neighbour-joining tree based on present in the Streptomyces. Our hypothesis partial 16S rRNA gene sequences (Figure that Streptomyces from marine ecosystem is a 4) showed relationships between GMY01, promising sources for novel NRPS.

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